CN109709253A - The method and kit of Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 - Google Patents
The method and kit of Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 Download PDFInfo
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Abstract
The present invention discloses the method and kit of Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12, take detection serum or detection blood plasma, be added methopterin in scalar quantity, by by free vitamin B12 from vitamin B12 separate out, cyano ion is provided, stablizing cobalamin isomers is cyanocobalamin；The step of vitamin B12 is enriched with using solid phase extraction, and the measurement for carrying out vitamin B12 in human plasma or serum is detected using tandem mass spectrum, high specificity, high sensitivity can meet the needs of clinical detection.
Description
Technical field
The present invention relates to vitamin detection field, in particular to a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12
Method and kit.
Background technique
Vitamin B12 is a kind of corrinoid (corrinoids) compound for containing corrin (corrin) ring, vitamin B12
Trivial name, which is called, does " cobalamin (cobalamin) ", is a kind of unique vitamin containing metallic element.However in human serum
About 1/3 vitamin B12 is not cobalamin, but other corrinoids, they are not have biology to human body in metabolism
It learns active.Mecobalamin element and 5 '-deoxyadenosyl cobalamins are main present in the active form and blood of vitamin B12
Form.
Human body cannot synthesize vitamin B12, rely primarily on animal food offer, and rich content person has hepatic and renal tissue, meat
Class, eggs and milk product.Vitamin B12 plays the role of very important in body metabolism, can be used as methyl-prop two
The coenzyme of sour list acyl coenzyme A isomerase, participates in the reaction that methylmalonyl-CoA is changed into mono succinate acyl coenzyme A, should
Reaction plays an important role in the metabolism of odd carbon fatty acid.Vitamin B12 can also be used as the change of Beta-methyl asparatate
The coenzyme of structure enzyme participates in the process that Beta-methyl asparatate is converted to glutamic acid.In addition, vitamin B12 cooperates with ginseng with folic acid
With transmethylation, intracorporal a variety of metabolic processes are influenced.When vitamin B12 deficiency, DNA dyssynthesis and huge children can lead to
Cell anemia influences neural myelin and is formed, to neurological symptom occur.
In addition, there are the binding protein of 3 kinds of vitamin B12s, i.e. TC I, TC II, TC III in human plasma
(transcobalamin I, II, III), under normal circumstances, about 80% or more Serum Vitamin B12 can be with III knot of TC I and TC
It closes, referred to as the full haptocorrin of serum, only 20% is combined with TC II, form holoTC II.The half-life period of haptocorrin is about entirely
240 hours, the half-life period of holoTC II was only 6 minutes.Therefore total serum vitamin B12 content can be used as a kind of main biology
Marker is horizontal for embodying body vitamin B12.
Currently, there are many detection methods of vitamin B12, including Chemiluminescence immunoassay, microbial method and isotope are put
Immunization method etc. is penetrated, however immunologic detection method and microbial process have many disadvantages, it is as follows:
The first, requirement of the immune response to reaction condition is more harsh, it is necessary to guarantee suitable pH, electrolyte environment and row
Except cross reaction；
Second, there are significant difference, that is, the accuracy in detection being immunoreacted obtains different vendor's detection kit testing result
Less than guarantee；
Third, poor specificity cannot effectively distinguish cobalamin and corrinoid, cannot provide accurate vitamin B12 content；
4th, radioimmunoassays there are radiation and pollution the problems such as.
Summary of the invention
It is an object of the invention to propose a kind of liquid chromatography tandem for defect existing for detection vitamin B12 at present
The method and kit of mass spectroscopy vitamin B12, high specificity, high sensitivity can meet the needs of clinical detection.
In order to realize that any of the above goal of the invention, the present invention provide Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12
Method, the vitamin B12 suitable for measurement serum or blood plasma, comprising the following steps:
(1) extraction and stabilization of vitamin B12:
The extraction of vitamin B12: by reference state vitamin B12 from cobalamin binding protein separate out；
The stabilization of vitamin B12: cyano ion is provided, (hydroxocobalamine, Mecobalamin element and 5- are de- for conversion cobalamin isomers
Oxygen adenosylcobalamin etc.) it is metastable cyanocobalamin；
(2) purification concentration cyanocobalamin；And
(3) tandem mass spectrum detects；
Wherein chromatography condition are as follows:
Chromatographic column:2.6μm Polar C18LC Column 50x 2.1mm；
Mobile phase A liquid: the ammonium acetate solution containing formic acid；
Mobile phase B liquid: the ammonium acetate methanol solution containing formic acid:
Flow velocity: 0.3-0.4mL/min；
Sample volume: 20 μ L-40 μ L；
Column temperature: 30-40 DEG C；
Wherein Mass Spectrometry Conditions are as follows:
Ion source: electric spray ion source；Scanning mode: positive ion mode；Ion spray voltage: 4500-5500V；Ion
Source temperature (TEM): 400-500 DEG C；Gas curtain gas (CUR): 20-35psi；Collision gas (CAD): 9psi；Ion source atomization gas
(GS1): 40-55psi；Ion source heating assists gas (GS2): 40-60psi；Scan pattern: MRM.
The detection ion pair and other conditions such as following table one of corresponding vitamin B12, table one:
In addition, in the present invention, using methopterin as interior scalar quantity, due to the color of methopterin and vitamin B12
It is very similar to compose retention behavior, therefore internal standard quantitation standard can be chosen as, at this point, the detection ion of corresponding methopterin and its
His condition such as table two, table two:
(4) quantitative detection:
Standard curve is made using vitamin B12 standard items:
Using concentration of standard solution as X-axis, the ratio of standard items and internal standard peak area is Y-axis, carries out linear regression analysis, warp
" I/X " weight obtains regression equation；
Testing result is obtained, regression equation is substituted into and obtains the content of B12 in serum or blood plasma.
In the extraction step of vitamin B12, have following several optional extractions when value:
1, using the disulfide bond in protein delivery agent reduction Vitamin B12 binding protein, vitamin B12 is destroyed using highly basic
With the hydrogen bond of combining unit and the coordinate bond of histidine thus by vitamin B12 separate out；
2, protein denaturation is controlled using hot conditions in buffer, so that vitamin B12 be combined from vitamin B12
Separate out in albumen；
3, using proteolytic enzyme protolysate, by vitamin B12 from Vitamin B12 binding protein separate out,
Wherein, in the first extracting mode, DTT (dithiothreitol dithio) or TCEP (three (2- carbonyls are may be selected in protein delivery agent
Base ethyl) microcosmic salt hydrochlorate), sodium oxide molybdena or potassium hydroxide may be selected in highly basic.
In second of extracting mode, sodium acetate buffer or citrate buffer is may be selected in buffer, protein-denatured
Condition selection are as follows: 100 DEG C of water-bath heating or 121 DEG C of autoclaves.
In the third extracting mode, pancreatic protease or papain is may be selected in protease.
In the stabilizing step of vitamin B12, using cyano ion by cobalamin isomers (hydroxocobalamine, Mecobalamin element and
5- deoxyadenosyl cobalamin etc.) it is converted into more stable cyanocobalamin, stable principle such as Fig. 2.Before chemo-immunity method
Unlike processing step, in an embodiment of the present invention, it is only necessary to provide cyano ion without providing chromogenic ion, because
It for testing principle of the present invention is detected by way of liquid chromatography mass, without using chemo-immunity method, therefore originally
The cyano ion of invention may be selected to be: with potassium cyanide or the complex solution of Cymag or cyano-containing, reduce sample pre-treatments
Difficulty and cost.
It is noted that the extraction of vitamin B12 synchronous can be carried out with the stabilizing step of vitamin B12, detection is improved
Efficiency.
In purification concentration cyanocobalamin step, two ways also may be selected and purified:
1, pass through HLB solid-phase extraction column desalination, purification, concentration.
2, selection vitamin B12 affine in immunity purification column purification concentration.
HLB refers to being gathered by a certain percentage by two kinds of monomers of lipophilicity divinylbenzene and hydrophily n-vinyl pyrrolidone
The specification condition of the macroporous copolymer of synthesis, selection is as follows: 1cc/30mg, producer are unlimited.Activation balance: 500-1000 μ L methanol
Activation, the ultrapure water balance of 500-1000 μ L.
Vitamin B12 affine in immunity decontaminating column uses the monoclonal antibody of high specific, and antibody has height to vitamin B12
Compatibility, when sample extracting solution passes through immune affinity column, vitamin B12 adds water elution nonbonding ingredient, adds in conjunction with antibody
Methanol elutes the vitamin B12 that antibody adsorbs.
In addition, the present invention provides a kind of kit of Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12, such as table three:
Compared with the prior art, the present invention has the following beneficial effects:
1, the method for measurement vitamin B12 provided by the invention and kit are using Liquid Chromatography-Tandem Mass Spectrometry detection human body
The content of interior vitamin B12 avoids being detected using immunization method, generates when so as to avoid immunization method detection various
Problem.
2, the kit and method for comparing detection food vitamins B12 have carried out purification concentration to cyanocobalamin, have been applicable in
The vitamin B12 of low concentration in detection serum and blood plasma.
3, this kit detection specificity is high, the concentration of accurate vitamin B12 can be obtained within a short period of time, in life
There is great application in analyte detection field.
Detailed description of the invention
Fig. 1 is the chemical structural drawing according to vitamin B12.
Fig. 2 is the method and reagent of the Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 of an embodiment according to the present invention
The vitamin B12 detection principle diagram of box.
Fig. 3 is the method and reagent of the Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 of an embodiment according to the present invention
The vitamin B12 quantitative criterion working curve of box.
The method and kit of the Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 of Fig. 4 embodiment according to the present invention
Vitamin B12 standard items chromatogram.
The method and kit of the Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 of Fig. 5 embodiment according to the present invention
Vitamin B12 internal standard methopterin chromatogram.
The method and kit of the Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 of Fig. 6 embodiment according to the present invention
Human serum sample in vitamin B12 chromatogram.
The method and reagent of the Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 of Fig. 7 according to another embodiment of the present invention
The chromatogram of vitamin B12 in the human serum sample of box.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art's every other embodiment obtained belong to what the present invention protected
Range.
It is understood that term " one " is interpreted as " at least one " or " one or more ", i.e., in one embodiment,
The quantity of one element can be one, and in a further embodiment, the quantity of the element can be it is multiple, term " one " is no
It can be interpreted as the limitation to quantity.
The present invention provides the feasibility that specific embodiment verifies method of the invention:
One, material:
The sample of 1.1 methodological studies experiment: serum, blood plasma pacify Laboratory of medical test from Hangzhou degree；
1.2 instrument
Nitrogen blows machine (MD200, Hangzhou Ao Sheng Instrument Ltd.)；96 orifice plates (U-shaped 0.2mL, Beaune Ai Jieer)；Centrifuge
(1-14K,SIGMA)；Centrifuge tube (1.5mL, AxyGen)；Chromatographic system, mass spectrometer system, (Waters Xevo TQD/I-Class
Triple level four bars mass spectrographs, Waters company)；
1.3 reagent consumptive materials:
HLB solid-phase extraction column, 1cc/30mg；Chromatographic column: Polar C18；(1mg/mL contains 2% hydroxide to potassium cyanide solution
Sodium, lark prestige)；Dithiothreitol (DTT) (1mol/L uses sodium acetate that pH5.2 concentration is 0.01mol/L as solution from preparing, Ah
Latin)；1%-2% glacial acetic acid solution；5% methanol；Ammonium acetate solution containing formic acid；Ammonium acetate methanol containing formic acid is molten
Liquid.
1.4 standard items: vitamin B12 standard items, (cyanocobalamin 100mg, Mike woods)；
1.5 internal standard compounds: 0.1 μ g/mL the methopterin, (first that 0.2% ammonium hydroxide methanol is 1mg/mL as solvent compound concentration
Then ammonia petrin stock solution is diluted to 0.1 μ g/mL with the ammonium acetate solution containing formic acid with methanol dilution to 10 μ g/mL,
Standard items are purchased from lark prestige)；
1.6 quality-control samples: (from preparing)；
Two, sample pre-treatments:
After sample pre-treatments, impurity is enriched with and removed to the vitamin B12 in reagent extracting solution with solid phase extraction,
High performance liquid chromatography tandem mass spectrum measurement, with methopterin for interior scalar quantity, it is fast that this method detects speed, and can be used for detecting people
Vitamin B12 in serum or blood plasma.
The preparation of standard solution: the cyanocobalamin methanol stock solution (containing 0.1% acetic acid) of 1mg/mL is prepared, with containing 0.1%
The methanol of acetic acid is diluted to 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 100ng/mL, 200ng/mL work respectively
Make stock solution, pipette the 10 above-mentioned working stock liquids of μ L respectively into 1.5mL centrifuge tube, be separately added into 190 μ LPBS buffers, mixes
It is even, it is bent that the standard that concentration is 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL is made
Line.
The preparation of quality-control sample: the cyanocobalamin methanol stock solution of 1mg/mL is diluted respectively with the methanol containing 0.1% acetic acid
To 4ng/mL, 12ng/mL, 24ng/mL, the 10 above-mentioned working stock liquids of μ L are pipetted respectively into 1.5mL centrifuge tube, are separately added into
190 μ LPBS buffers mix, and the quality-control sample that concentration is 0.2ng/mL, 0.6ng/mL, 1.2ng/mL is made.
Embodiment 1:
Sample pre-treatments:
Precision measures standard solution, and quality-control sample or human serum are placed in clean centrifuge tube；Added with the volume ratio of 10:1
Enter internal standard (0.1 μ g/mL methopterin), it is molten that target potassium cyanide in 2.5-4 volume times (1mg/mL contains 2% sodium hydroxide) is added
Liquid is added target dithiothreitol (DTT) solution in 0.5 volume times, mixes 3-5 minutes, be protected from light 15-30 minutes ultrasonic；
It is added in 1%-2% glacial acetic acid solution and sample, mixing 2 minutes, 12000rpm/min is centrifuged 5 minutes.Take supernatant
Liquid loading extremely activates the HLB solid-phase extraction column balanced, and after sample outflow, the washing of 5% methanol is miscellaneous, and dry 1-2min uses first
Alcohol elutes in two times, and nitrogen is blown, and redissolves, and is vortexed and mixes 5min, pipettes to 96 orifice plates, is centrifuged 5min, sample introduction LC-MS-MS.
In addition, eluting in two times if it is to be changed to 50% methanol-water using centrifuge concentrator, 50% methanol water elution is wanted
It after being concentrated to dryness with centrifuge concentrator, redissolves, is vortexed and mixes 5min, pipette to 96 orifice plates, be centrifuged 5min, sample introduction LC-MS-MS.
Specifically, accurate measure standard solution, quality-control sample or 200 μ L of human serum are placed in clean centrifuge tube；It is added
50 μ L-200 μ L potassium cyanide (1mg/mL contains 2% sodium hydroxide) solution or one is added in internal standard (0.1 μ g/mL methopterin) 20 μ L
Determine the complex compound containing cyanogen root of concentration, 10 μ L dithiothreitol (DTT) solution are added, mixes 3-5 minutes, be protected from light 15-30 minutes ultrasonic.
It is added in 1%-2% glacial acetic acid solution and sample, mixing 2 minutes, 12000rpm/min is centrifuged 5 minutes.Take supernatant loading extremely
The HLB solid-phase extraction column balanced is activated, after sample outflow, is separately added into 1mL ultrapure water (this portion can also be omitted), 1mL
The washing of 5% methanol is miscellaneous, dry 1-2min (it is pay attention to removing moisture remaining on solid-phase extraction column clean, to subsequent nitrogen blow when
Between influence it is very big), 600 μ L methanol (if there is centrifuge concentrator will be changed to 50% methanol water elution) elute in two times, and nitrogen is blown
(50% methanol water elution is concentrated to dryness with centrifuge concentrator) redissolves, is vortexed and mixes 5min, pipettes to 96 orifice plates, centrifugation
5min, sample introduction LC-MS-MS.
Embodiment 2:
Sample pre-treatments: precision measures standard solution, and quality-control sample or human plasma are placed in clean centrifuge tube；With 10:1
Volume ratio internal standard (0.1 μ g/mL methopterin) is added, be added in 40-60 volume times target 0.1M/L pH4.0-4.6 it
Between sodium acetate buffer (Cymag containing a certain concentration), mix 3-5 minute, 100 DEG C water-bath 30 minutes, be put in ice bath cool down,
12000rpm/min is centrifuged 5 minutes.
It takes supernatant loading extremely to activate the HLB solid-phase extraction column balanced, after sample outflow, is separately added into ultrapure water
(can also omit this step), the washing of 5% methanol is miscellaneous, and dry 1-2min (pays attention to removing moisture remaining on solid-phase extraction column dry
Only, the time effects blown to subsequent nitrogen are very big), it is eluted in two times with methanol, nitrogen is blown, and is redissolved, and is vortexed and is mixed 5min, pipettes to 96
Orifice plate is centrifuged 5min, sample introduction LC-MS-MS.
Specifically, accurate measure standard solution, quality-control sample or 200 μ L of human serum are placed in clean centrifuge tube；It is added
The acetate buffer between 800 μ L-1200 μ L 0.1M/L pH4.0-4.6 is added in internal standard (0.1 μ g/mL methopterin) 20 μ L
Liquid (Cymag containing a certain concentration), mix 3-5 minute, 100 DEG C water-bath 30 minutes, be put in ice bath cool down.12000rpm/min from
The heart 5 minutes.It takes supernatant loading extremely to activate the HLB solid-phase extraction column balanced, after sample outflow, it is ultrapure to be separately added into 1mL
Water (can also omit this portion), and the washing of 5% methanol of 1mL is miscellaneous, and dry 1-2min (pays attention to moisture remaining on solid-phase extraction column is clear
Except clean, the time effects blown subsequent nitrogen are very big), 600 μ L methanol are (if there is centrifuge concentrator will be changed to the washing of 50% methanol
It is de-) it elutes in two times, nitrogen is blown (50% methanol water elution is concentrated to dryness with centrifuge concentrator), is redissolved, and is vortexed and is mixed 5min, is moved
It takes to 96 orifice plates, is centrifuged 5min, sample introduction LC-MS-MS.
Three, Liquid Chromatography-Tandem Mass Spectrometry detects:
Chromatographic condition:
Chromatographic column: Polar C18；Mobile phase: A liquid: the ammonium acetate solution containing formic acid, B liquid: the acetic acid containing formic acid
Ammonium methanol solution；Flow velocity: 0.4mL/min, column temperature are 35 DEG C, 20 μ L of sample introduction；Isocratic elution.
Mass Spectrometry Conditions:
Ion source: electric spray ion source；Scanning mode: positive ion mode；Ion spray voltage: 5500V；Ion source temperature
(TEM): 500 DEG C；Gas curtain gas (CUR): 20psi；Collision gas (CAD): 9psi；Ion source atomization gas (GS1): 55psi；Ion source
Heating auxiliary gas (GS2): 60psi；Scan pattern: MRM.
Vitamin B12 and the setting of interior target MRM method
Four, data processing:
4.1 obtain the chromatogram of vitamin standard items
The chromatogram of vitamin B12 standard items is obtained as shown in figure 4, lower limit of quantitation as standard curve.
4.2 obtain the chromatogram of vitamin B12 internal standard internal standard methopterin.
The chromatogram of vitamin B12 internal standard methopterin is obtained as shown in figure 5, its chromatographic peak retention time and vitamin
B12 is close, can be very good the fluctuation that in calibration sample pretreatment process and chromatographic separation process generates, and improves analysis result
Accuracy.
4.3 obtain the quantitative criterion working curve of vitamin B12:
The vitamin B12 standard curve for obtaining various concentration is measured, and is obtained detection data, is with concentration of standard solution
The ratio of X-axis, standard and internal standard peak area is Y-axis, carries out linear regression analysis, obtains regression equation through " 1/X " weight: y=
0.01672x+0.099974, linear coefficient 0.99974, as shown in figure 3, linear fit equation, linear good, it is fixed to meet
Amount requires.
Five, testing result:
5.1 testing results 1:
It is detected with the sample that embodiment 1 is prepared, the chromatogram detected such as Fig. 6, it will be appreciated from fig. 6 that chromatography
Peak shape is preferable, and retention time is corresponding with vitamin B12 standard items, measured value 0.41ng/mL.
5.2 testing results 2:
It is detected with the sample that embodiment 2 is prepared, the chromatogram detected such as Fig. 7, as shown in Figure 7, chromatography
Peak shape is preferable, and retention time is corresponding with vitamin B12 standard items, measured value 0.59ng/mL.
Six, the feasibility detection of this method:
6.1, detection limit:
The vitamin B12 standard solution of 0.03ng/mL is prepared, upper instrument is measured, and replication 3 times, calculates noise
Than mean value, result 4.This method instrument detection limit is 0.03ng/mL.
6.2 precision:
Batch interior betweenrun precision degree of 0.2ng/mL, 0.6ng/mL, 1.2ng/mL concentration meets clinic within 15%
Detect the requirement to precision.
6.3 accuracy:
0.2ng/mL, 0.6ng/mL, 1.2ng/mL concentration batch in batch between accuracy quality-control sample sign value ±
Within 15%, meet requirement of the clinical detection to accuracy.
Seven, the kit of Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12:
The present invention is not limited to above-mentioned preferred forms, anyone can show that other are various under the inspiration of the present invention
The product of form, however, make any variation in its shape or structure, it is all that there is skill identical or similar to the present application
Art scheme, is within the scope of the present invention.
Claims (10)
1. the method that Liquid Chromatography-Tandem Mass Spectrometry measures vitamin B12, measures vitamin B12 in serum or blood plasma, feature exists
In, comprising:
(1) detection serum or detection blood plasma are taken, methopterin internal standard is added；
(2) extraction and stabilization of vitamin B12:
The extraction of vitamin B12: by reference state vitamin B12 from cobalamin binding protein separate out；
The stabilization of vitamin B12: cyano ion, conversion cobalamin isomers (hydroxocobalamine, Mecobalamin element and 5- deoxidation gland are provided
Glycosides cobalamin) it is metastable cyanocobalamin；
(3) purification concentration cyanocobalamin:
Purification enrichment is carried out to vitamin B12 using solid phase extraction；And
(4) tandem mass spectrum detects；
Wherein chromatography condition are as follows:
Chromatographic column: 2.6 μm of Polar C18 of KinetexLC Column 50 x 2.1mm；
Mobile phase A liquid: the ammonium acetate solution containing formic acid；
Mobile phase B liquid: the ammonium acetate methanol solution containing formic acid:
Flow velocity: 0.3-0.4mL/min；
Sample volume: 20 μ L-40 μ L；
Column temperature: 30-40 DEG C；
Wherein Mass Spectrometry Conditions are as follows:
Ion source: electric spray ion source；Scanning mode: positive ion mode；Ion spray voltage: 4500-5500V；Ion source temperature
Degree: 400-500 DEG C；Gas curtain gas: 20-35psi；Collision gas: 9psi；Ion source atomization gas: 40-55psi；Ion source heating auxiliary
Gas: 40-60psi；Scan pattern: MRM；
Corresponding vitamin B12 and interior target detection ion pair and other conditions are as follows:
(4) quantitative detection:
Testing result is obtained, vitamin B12 standard items regression equation is substituted into and obtains the content of B12 in serum or blood plasma, wherein tieing up
For raw element B12 standard items regression equation using concentration of standard solution as X-axis, the ratio of standard items and internal standard peak area is Y-axis, carries out line
Property regression analysis obtains.
2. the method for Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 according to claim 1, which is characterized in that described
Chromatographic condition are as follows: flow velocity: 0.4mL/min, column temperature are 35 DEG C, 20 μ L of sample introduction；Gradient elution, the Mass Spectrometry Conditions are as follows: ion
Spray voltage: 5500V；Ion source temperature: 500 DEG C；Gas curtain gas: 20psi；Collision gas: 9psi；Ion source atomization gas: 55psi；
Ion source heating assists gas: 60psi；Scan pattern: MRM.
3. the method for Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 according to claim 1, the vitamin B12
In stabilizing step, the complex solution of the cyano ion selection potassium cyanide or Cymag or cyano-containing that provide.
4. the method for Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 according to claim 1, which is characterized in that selection
Pass through HLB solid-phase extraction column desalination, purification, concentration；Condition control are as follows: activation balance: the activation of 500-1000 μ L methanol, 500-
The 1000 ultrapure water balances of μ L.
5. the method for Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 according to claim 1, which is characterized in that described
The extraction of vitamin B12 selects one of following three kinds of methods:
Using the disulfide bond in protein delivery agent reduction Vitamin B12 binding protein, vitamin B12 and combination are destroyed using highly basic
The hydrogen bond of unit and the coordinate bond of histidine；
Protein denaturation is controlled using hot conditions in buffer；
Using proteolytic enzyme protolysate, by vitamin B12 from Vitamin B12 binding protein separate out；
Wherein the protein delivery agent is selected from one kind of dithiothreitol dithio or three (2- carbonylethyl) microcosmic salt hydrochlorates；The buffering
Liquid is selected from sodium acetate buffer or citrate buffer, protein-denatured condition selection are as follows: 100 DEG C of water-bath heating or 121 DEG C
Autoclave；The protease is selected from pancreatic protease or papain.
6. the method for Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 according to claim 5, which is characterized in that measure 1
The detection serum or detection blood plasma of parts by volume are placed in clean centrifuge tube；The methopterin of 0.1 parts by volume, 0.1 μ g/mL is added, adds
Enter the potassium cyanide solution of 0.4-1 parts by volume, wherein 2% sodium hydroxide that the potassium cyanide solution is 1mg/mL containing concentration, is added
The dithiothreitol (DTT) solution of 0.05 parts by volume is mixed 3-5 minutes, is protected from light 15-30 minutes ultrasonic；It is molten that 1%-2% glacial acetic acid is added
It is centrifuged 5 minutes in liquid with sample, mixing 2 minutes, 12000rpm/min, supernatant loading is taken extremely to activate the HLB solid phase balanced
Extraction column, after sample outflow, the washing of 5% methanol is miscellaneous, and dry 1-2min is eluted in two times with methanol, and nitrogen is blown, and is redissolved, and is vortexed
5min is mixed, pipettes to 96 orifice plates, is centrifuged 5min.
7. the method for Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 according to claim 5, which is characterized in that measure 1
The detection serum or detection blood plasma of parts by volume are placed in clean centrifuge tube；4- is added in the methopterin of 0.1 parts by volume, 0.1 μ g/mL
The sodium acetate buffer of containing a certain concentration Cymag of the pH of target 0.1M/L between 4.0-4.6 in 6 parts by volume mixes 3-5
Minute, 100 DEG C water-bath 30 minutes, be put in that ice bath is cooling, and 12000rpm/min is centrifuged 5 minutes, take supernatant loading flat to activating
The HLB solid-phase extraction column to have weighed, after sample outflow, after sample outflow, the washing of 5% methanol is miscellaneous, and dry 1-2min uses methanol
It elutes in two times, nitrogen is blown, and is redissolved, and is vortexed and is mixed 5min, is pipetted to 96 orifice plates, is centrifuged 5min.
8. according to the method for any Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 of claim 6-7, feature exists
In being changed to 50% methanol-water using centrifuge concentrator and elute in two times, 50% methanol water elution is concentrated with centrifuge concentrator
It to after dry, redissolves, is vortexed and mixes 5min, pipette to 96 orifice plates, be centrifuged 5min.
9. the method for Liquid Chromatography-Tandem Mass Spectrometry measurement vitamin B12 according to claim 1, which is characterized in that standard
The detecting step that product solution and quality-control product are same as test sample is detected, the concentration selection of standard solution are as follows: 0.1ng/mL-
10ng/mL；
The configuration of the standard solution: preparing the cyanocobalamin methanol stock solution (containing 0.1% acetic acid) of 1mg/mL, with containing
The methanol of 0.1% acetic acid is diluted to the working stock liquid of 2ng/mL-200ng/mL respectively, pipettes the above-mentioned work of 1 parts by volume respectively
Make stock solution into 1.5mL centrifuge tube, be separately added into the μ LPBS buffer of 19 parts by volume, mix, it is 0.1ng/ that concentration, which is made,
The standard curve of mL-10ng/mL.
10. the kit of phase chromatographic tandem mass spectroscopy vitamin B12 characterized by comprising
The method that the kit measures vitamin B12 using the Liquid Chromatography-Tandem Mass Spectrometry as described in claims 1 to 9 is any,
Measure vitamin B12 in serum or blood plasma.
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| CN111398439A (en) * | 2020-03-06 | 2020-07-10 | 权丽 | Liquid chromatography-tandem mass spectrometry detection method for B vitamins in serum |
| CN111624074A (en) * | 2020-07-07 | 2020-09-04 | 江苏拜明生物技术有限公司 | Release agent for detecting content of vitamin B12 in serum, preparation and application |
| CN112697936A (en) * | 2020-12-18 | 2021-04-23 | 卓和药业集团有限公司 | Method for determining substances related to mecobalamin particles |
| CN113009013A (en) * | 2021-02-23 | 2021-06-22 | 北京斯利安药业有限公司 | Method for analyzing impurities in mecobalamin sample |
| CN113138236A (en) * | 2021-03-31 | 2021-07-20 | 山东英盛生物技术有限公司 | Method for quantitatively detecting vitamin B12 in serum and pretreatment kit |
| CN113189252A (en) * | 2021-04-19 | 2021-07-30 | 中国医学科学院北京协和医院 | Detection method of total cobalamins and cyanocobalamins, detection kit and application thereof |
| CN113984943A (en) * | 2021-10-29 | 2022-01-28 | 哈尔滨医科大学 | Method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum |
| CN114011111A (en) * | 2022-01-05 | 2022-02-08 | 广州科方生物技术股份有限公司 | Extraction liquid for extracting vitamins from binding protein and preparation method and application thereof |
| CN114216982A (en) * | 2021-12-14 | 2022-03-22 | 黑龙江飞鹤乳业有限公司 | Vitamin B12Is detected by |
| CN116990423A (en) * | 2023-07-14 | 2023-11-03 | 河北伊莱莎生物技术有限公司 | Vitamin B12 quantitative detection method and method for rapidly drawing standard curve |
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| CN111398439A (en) * | 2020-03-06 | 2020-07-10 | 权丽 | Liquid chromatography-tandem mass spectrometry detection method for B vitamins in serum |
| CN111624074A (en) * | 2020-07-07 | 2020-09-04 | 江苏拜明生物技术有限公司 | Release agent for detecting content of vitamin B12 in serum, preparation and application |
| CN111624074B (en) * | 2020-07-07 | 2024-01-30 | 江苏拜明生物技术有限公司 | Release agent for detecting vitamin B12 content in serum, preparation and application |
| CN112697936A (en) * | 2020-12-18 | 2021-04-23 | 卓和药业集团有限公司 | Method for determining substances related to mecobalamin particles |
| CN113009013A (en) * | 2021-02-23 | 2021-06-22 | 北京斯利安药业有限公司 | Method for analyzing impurities in mecobalamin sample |
| CN113138236A (en) * | 2021-03-31 | 2021-07-20 | 山东英盛生物技术有限公司 | Method for quantitatively detecting vitamin B12 in serum and pretreatment kit |
| CN113189252A (en) * | 2021-04-19 | 2021-07-30 | 中国医学科学院北京协和医院 | Detection method of total cobalamins and cyanocobalamins, detection kit and application thereof |
| CN113984943A (en) * | 2021-10-29 | 2022-01-28 | 哈尔滨医科大学 | Method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum |
| CN113984943B (en) * | 2021-10-29 | 2023-12-22 | 哈尔滨医科大学 | Method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum |
| CN114216982A (en) * | 2021-12-14 | 2022-03-22 | 黑龙江飞鹤乳业有限公司 | Vitamin B12Is detected by |
| CN114011111A (en) * | 2022-01-05 | 2022-02-08 | 广州科方生物技术股份有限公司 | Extraction liquid for extracting vitamins from binding protein and preparation method and application thereof |
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